The synaptic vesicle uptake of classical transmitters depends on a H+ electrochemical driving force (?H+), and generally involves the exchange of cytosolic transmitter for lumenal H+. However, vesicular glutamate transport relies almost entirely on the electrical component of this gradient (??) rather than the pH gradient (?pH), and undergoes unusual, allosteric regulation by H+ and Cl-. The vesicular glutamate transporters (VGLUTs) also exhibit an associated Cl- conductance, and the physiological role of these properties remains unknown. Further, the VGLUTs belong to the solute carrier 17 (SLC17) family which includes other members that rely on ?pH rather than ?? for transport in the opposite direction from VGLUTs. The long-term objective of this proposal is to understand how the properties of vesicular glutamate transport contribute to synaptic transmission. The strategy uses structure to identify the mechanisms common to all family members and understand how their adaptation confers the specific properties of vesicular glutamate transport. We have determined the first structures of an SLC17 family member, E. coli D-galactonate transporter DgoT, which is closely related in sequence to the VGLUTs. DgoT contains a polar pocket within the N-terminal lobe connected to the periplasm through a putative H+ tunnel evident in the inwardly oriented structure. An outwardly oriented structure contains galactonate occluded in the substrate recognition site. The structures predict that delivery of periplasmic H+ to a glutamate in transmembrane domain (TM) 4 liberates an interacting arginine in TM1 to bind substrate. In contrast to the VGLUTs but like other SLC17 proteins, DgoT catalyzes H+ cotransport. Although the critical residues are conserved to the VGLUTs, they thus serve a different function in DgoT. We will now 1) Elucidate the mechanism that couples transport of galactonate to H+ in DgoT. Using assays for exchange and binding as well as net uptake, we will determine how protonation of DgoT contributes to substrate recognition. 2) Determine the structural basis for vesicular glutamate transport. We will use a combination of crystallography and cryo-electron microscopy to determine the structure of a VGLUT. 3) Elucidate the mechanisms responsible for allosteric regulation of the VGLUTs. We will leverage the structures as well as the available assays for both DgoT and the mammalian proteins to understand the allosteric regulation of VGLUTs by H+ and Cl-. We will also use electrophysiology to assess a channel suggested by the structure, and determine its relationship to glutamate flux.